JPH03276067A - Composite device for measuring substance in liquid - Google Patents

Abstract

PURPOSE: To obtain a composite device for measuring substance in liquid which can be used at a site or in the field where an objective substance is present by passing through at least first and second porous material layers abutting each other, one or more material receiving region in the device, the second layer and a liquid channel therein. CONSTITUTION: In a double layer device 10, a first upper porous material layer 20 has a sample port 16 while a second lower porous material layer 11 has a discharge port 19, and communicating with the member 11 through an interface 14. The sample port 16 is surrounded by a liquid barrier means 13 and the members 20, 11 may be made of same material or not, may have identical porosity or not and/or may have identical sucking action or not. Consequently, a portable device is obtained wherein a visible color change takes place substantially upon throwing in of an inspection sample and a measurement can be read out remotely with eyes without requiring instrumental measurement.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a diagnostic test device for determining the presence of a substance in a liquid medium, and more particularly to a diagnostic test device for determining the presence of substances in liquid media, particularly in biological and industrial applications. related to, or related to, immunological, live provision in liquids related to agriculture. Still more particularly, the present invention relates to the provision of a multilayer device capable of rapidly detecting such substances and measuring the results without a reading device.

BACKGROUND OF THE INVENTION The field of diagnostic testing has evolved from the use of complex radioimmunoassays and enzyme immunoassays to the use of single-card devices, which are typically analyzed under field conditions. There has been a need to improve the readability of such devices without the need for equipment and to make the devices readable in a short time in order to be able to measure target substances. Attempts have also been made to reduce the number of steps required to perform a test by introducing an element of security.

A test device that is portable, produces a visible color change almost immediately upon introduction of the test sample, and can be read by the naked eye at a remote location without the need for instrument measurements would be of great benefit.

It would be beneficial to have a system that did not require a large number of steps to perform the test, especially by eliminating the large number of washing steps and long incubation times, which would dramatically increase the time and convenience of reading test results. Will.

(Problems to be Solved by the Invention) One object of the present invention is to provide a testing device that can be used in a place where a substance to be detected is present, such as on-site or outdoors.

A further object of the present invention is to provide a diagnostic testing device which, after addition of a sample, can generate a reaction that makes the final result visible, with little or no additional effort. That's true.

A further object of the present invention is to provide a test device comprising a filter-type planar membrane having multiple layers and containing binding substances such as antibodies and/or antigens in appropriate areas to promote the reaction mechanism. That's true. Other objects and advantages will become apparent from the following description and accompanying drawings.

SUMMARY OF THE INVENTION The present invention provides a composite structure of porous materials that allows liquid flow by selecting various pore sizes and juxtaposing liquid barrier means within appropriate areas of the structure. The objective is to provide a composite structure that, when channeled and selected with appropriate materials, provides immunologically unexpected fluid flow forces.

Problems commonly encountered in the immunoassay field arise when performing competitive or sandwich-type binding assays on solid porous matrices. Immunological reactions in such methods often do not occur sufficiently quickly or appreciably without several washing steps and addition of reagents. Even with additional steps, the response often does not become noticeable to the naked eye in a useful time.

In a typical competitive binding assay, an externally supplied labeled antigen competes with the sample antigen and reacts with an externally supplied antibody (if the analyte is an antibody, a suitable binding partner is selected). Each labeled antigen molecule or antigen molecules present in the sample has the opportunity to react with the supplied antibody, and their degree of reactivity is a measure of the antigen concentration in the original sample.

Such systems are useful in the present invention.

In theory, competitive measurements can be performed by contacting a labeled antigen/antibody complex with the antigen of the sample and displacing the labeled antigen from the complex.

That is, if a labeled antigen/antibody complex is immobilized on a solid substrate and the complex is brought into contact with an unlabeled antigen, displacement of the labeled antigen is expected to occur. Unfortunately, when the solid substrate is a porous material, mere contact as described above does not always result in adequate displacement for detection of the analyte on a commercial basis. In a simple, one-step procedure, the inventors have demonstrated the ability to selectively displace labeled antigen from a labeled antigen/antibody complex by a sample antigen (if the complex was originally formed and thus provided) or by displacing the labeled antigen from the sample to be analyzed. immobilized capture binding partner
They have discovered structures and techniques that create effective competition between labeled analytes supplied externally as capture binding partners and rapidly visualize such reactions.

Other forms of immunoassays, such as the Sand-Deutsche method or competitive binding method using the analyte detection system when the analyte is an antibody rather than the aforementioned antigen, can also be used as shown in the detailed description of the invention below. be.

In short, this point has nothing to do with the attached drawings.
The purpose of the present invention is to provide a structure with various pore sizes and liquid flow barriers so that a sample can be quickly sucked up in a predetermined clear flow direction to a reaction area where a desired reaction occurs.

In its most general form, the present invention contemplates a structure having two planar flat porous members in close contact with each other on their planar surfaces. The multilayer device is configured to: - place or form a small volume of liquid test sample, such as a droplet, on or within a sample receiving area provided in the top member and adjacent to the sample receiving area; The presence of a liquid flow barrier forces the sample liquid to traverse
nsversely) to reach the bottom member. This sample-receiving area may be a hole in the top member, or it may be an appropriately shaped or limited portion of the top member itself, taking into account the type of test being performed.

The device delivers a flow of sample fluid from the sample receiving port or from an area along the predetermined flow path to the bottom member, depending on a preswaged and selected flow path, and ultimately to the bottom member. Alternatively, it may be configured to force flow into the reaction area on the top member.

The sample fluid does not flow laterally through the sample receiving area;
The flow crosses from the sample receiving area in or on the top member to the second member and finally reaches the reaction area by cross-flow to the top member. Typically, the reaction zone is located on the top member, but under appropriate circumstances it may be located on the bottom member.

The properties of the members include the rapid tensile action of the top member and/or
or the differential wicking (diff) between the bottom member and the top member.
It can be modified to exhibit a pumping action due to its erential wicking characteristics. The flow of the sample within the second member receiving the sample from the sample receiving area also includes:
The flow path within the second member is constrained by a barrier means which forces the flow path within a defined space and thus the direction of flow to the first member.

This action activates a wide range of possible reactions and reaction sites. For example, if the sample is forced not to flow substantially laterally across the top member, but transverse flow is forced, then the analyte of the sample dropped onto the top member will be It can eventually be moved to a reaction area located on the top member. This reaction zone (located on the top member or on the bottom member) may have the sample deposited thereon, or may itself be guided by additional reagents added directly or by the same route as the original sample deposit. The storage area serves as a storage area for additional reagents. The present invention involves arranging a defined sample receiving area alongside some type of liquid flow barrier and positioning the reaction area in a predetermined manner so as to direct the sample liquid flow in a preselected manner. It is necessary.

The devices of the invention have a very wide variety.

For example, the member itself may be either hydrophilic or hydrophobic depending on the nature of the test and the analyte being tested. for example,
By appropriately selecting the structure and composition of the components, analysis of aqueous or non-aqueous samples can be performed. Non-aqueous samples containing organic solvent-based materials can be used with hydrophobic members under appropriate circumstances. If desired, the present invention also allows for the use of hydrophobic materials in aqueous samples. Examples of hydrophobic materials are glass fibers, certain nylons, Teflon and polyvinyl chloride. Examples of hydrophilic materials are paper, certain cellulose acetates, polyvinylidene fluoride, cellulose nitrate, polypropylene, certain microglass fiber compositions, and the like.

Combining hydrophilic or hydrophobic properties with components of varying porosities and bonding properties allows a) to be performed on both aqueous and non-aqueous samples, and b) to work with one or both components. Binding of reactants at various regions in the top and sample or reagent flow paths;
tial control) is given to the device.

In one form of immunoassay, the sample is added to the sample port and the reactants are hydrated in the bottom or underlying member.

The reactant may be an enzyme or an indicator such as colloidal gold.
a competitive analyte bound to (cator).

The binding (capture) antibody is in this case located in the reaction area. This member may be protein-binding if retention of the capture antibody is desired. The lower layer member may be non-protein binding in the area where the indicator conjugate is located, but at the end after the reaction area, it retains the reactant and allows it to enter the reaction area. It can be made protein-binding to prevent return due to diffusion.

In another assay, the bottom member is initially made protein-binding;
During fabrication of the device, one or more measuring enzymes can be attached to it to determine the presence of a substrate in a sample solution. The sample is sequentially moved in a predetermined manner through the lower member in contact with a specific enzyme to perform a production reaction, and the product is observed in the reaction zone.

It will be apparent to those skilled in the art that numerous variations of immunoassays can be performed with this device. These variations include, but are not limited to, competitive assays for low volume analytes, sandwich assays, and direct detection of sample reactants. It is also clear that this device can be used to perform other non-immunological assays such as substrate and product detection, nucleic acid probes, lectins or other Gantt-receptor pairs in conjunction with various indicator systems. Probably.

To further illustrate, when a liquid sample is added to the sample area in or above the upper member, the liquid is drawn up into the lower member by capillary action. The speed is related to the properties of the selected member. Under certain circumstances, it may be desirable to have the pore size of the lower member substantially larger than the pore size of the upper member to facilitate pumping of return flow from the bottom member to the top member.

The edges of the sample area are made impermeable by the provision of barrier means so that the sample is restricted from moving laterally out of the sample receiving area. These barrier means include compressed areas of material, slots, discontinuities, sonic or thermoformed barriers, and the like, all known in the art.

Once the liquid enters the lower member, it moves laterally and transversely, and a juxtaposed unit located within the lower member directs the liquid flow back up to the top member and into contact with the reagents, etc. in the reaction zone. Comes into contact with the permeation barrier.

The reaction zone may include an indicator system element useful for detecting the analyte (or substrate) of interest. If desired, the reaction zone may additionally be disposed within the lower member and the top member disposed within the reaction zone. A remote area may be provided for the accumulation of reactants and samples that have passed through the area.

For example, in a preferred embodiment for carrying out a competitive immunoassay, the reaction region contains an antibody directed against the analyte of interest (capture antibody), said antibody being covalently or otherwise bound to the overlying member. It is convenient. If desired in this regard, a protein-bound member may be selected as the top member;
Antibody binding may be promoted. A conjugate is selected that has an indicator molecule attached to the analyte of interest. Suitable conjugates are those in which the analyte is bound directly to colloidal gold, if possible, or, if the analyte is unable or poorly bound to gold, the analyte is bound to a carrier molecule. Colloidal gold is a well-known reagent for diagnostics because of its characteristic reddish color. As carrier molecules, use may be made, for example, of natural or synthetic proteins or other macromolecules, such as BSA, poly-L-lysine, polysaccharides, histones, casein, horseradish peroxidase, and the like. The consinate may be mixed with the sample prior to addition to the receiving zone, or it may be placed somewhere within a predetermined liquid flow path within the device prior to the reaction zone. If the analyte in the sample is homologous to the analyte attached to the colloidal gold, it will compete with the analyte-gold conjugate in the reaction zone for the capture antibody.

If the concentration of antigen/analyte-gold conjugate is chosen appropriately, taking into account the measurement conditions, the analyte-gold conjugate will outcompete with the analyte in the sample and bind to the capture antibody. There will be no remaining condisgates. The reaction is confirmed by the absence of gold accumulation in the reaction zone. Thus, a positive reaction is indicated by the absence of a color change in the reaction zone (i.e., absence of consequents).

This reaction is commonly referred to as a competitive binding assay and is typical of what is performed in the devices of the present invention. Other forms can also be used as well as those described below.

The following is a brief description of the drawings, which are presented in consideration for a better understanding of the invention.

FIG. 1 is a cross-sectional view of the device of the present invention taken along the plane AA in FIG. 2. FIG. 2 is an embodiment of the device of the invention shown in a circular shape. FIG. 3 shows the device of the present invention after the test liquid has been manipulated and acted upon in the device of FIG.

FIG. 4 is a top view of another embodiment of the device of the invention.

FIG. 1 shows a two-layer device 10 according to the present invention, taken along the line AA in FIG. Two of the four reaction zones are shown. Upper layer (first porous material layer) 2
0 has a sample opening (sample receiving area) 16, and the lower layer 11 (second porous material layer) 20 and the member 11 may be made of the same or different materials, and have the same or different porous holes. and/or have the same or different wicking action. In one preferred embodiment of the invention,
The pore size of the bottom layer is about 5 to 10 times the pore size of the top layer member, and the pore size of the top layer member is in the range of 0.2-0.75 microns.

Regarding the size of the pores, even if they are large, they should not be so large that liquid will accumulate in them. This is because if the pores are too large and in such a state, the desired pumping action cannot be obtained. A highly desirable pore size is one that "pushes" the sample through the sample port into the lower member and subsequently (as explained below) draws the liquid back from the lower member into the first member.

Members 11 and 20 are barrier (liquid barrier means) 13a
and 13b, and these barriers are typically added during the manufacturing process by welding or by forming slots, discontinuities, etc.

Barrier 13b is formed to provide additional glue reservoir room and is an optional element depending on the desired glue reservoir size. Barrier 13c is also an optional element; in fact, barrier 13 alone is usually sufficient. However, due to test dynamics and device diameter considerations, it may be desirable to direct the ascending liquid flow into the top member at a point slightly forward of the sample receiving area. In such cases,
A barrier 13c is provided and the interface 14a is made impermeable or permeable as desired.

On the member 20 is a reaction zone 15 containing various reactants arranged according to and with consideration for the final test to be performed. As shown in Figure 2, these reaction areas are divided into two
There are two or more locations, and they are used for the same or different analysis targets from the same sample, or for controls. Although the device IO may have antibodies or antigens, for the purposes of this discussion it is preferred that the antibody 17 be bound to the member 10 by a covalent bond or other physical or chemical bond.

The member 11 is provided with a colloidal gold conjugate 18 designated Au- or other indicator system conjugated to an antigen specific for and reactive with the antibody 17. That is, the completed, ready-to-use complex form has a conjugate 18 of gold (or other indicator system) to antigen that is loaded into the device flow path in front of the reaction zone 15. Alternatively, instead of loading the conjugate into the device, it may be mixed with the sample and the sample/conjugate mixture flowed into the flow path leading to the reaction zone 15.

Instead of gold, enzymes, fluorescent agents, latex beads, luciferase,
Other detection systems such as chemiluminescent agents are used in immunoassays.

In use, a liquid sample is dropped into the sample port 16 and the sample diffuses into the member 11 to mix with the conjugate 18.

Lateral flow is impeded by barrier 13, which directs the flow of sample to the second member. Upon reaching the second member, the liquid is directed laterally until it reaches a barrier (e.g. 13a) or no further;
It is mixed with conjugate 18 and "pulled" into member 20. This mixture then moves to reaction zone 15. In reaction zone 15, if the sample contains an analyte corresponding to the antigen conjugated to the indicator system, it will compete with the conjugate for reaction with antibody 17. If the sample does not contain such an antigen, the only reactant in reaction area 15, other than antibody 17, will be the conjugate, which will react and exhibit a color change. If the sample contains the relevant antigen, the reaction at 15 will be largely due to the presence of such antigen. This is because the concentrations of consequate and antibody 17 selected during device fabrication are carefully adjusted to react well with the commonly encountered concentrations of the sample analyte.

That is, positive results are shown with no color change. For example, in FIG. 3, such results are indicated by "S" for the sample bound to antibody 17. This gold/antigen complex reaches members 20 and 11. If desired, additional reservoir chambers may be provided in member 11 depending on how much reservoir space is required as a function of the test being performed. Larger capacity reservoir-type members are easily provided by expanding the member 20 laterally. Additionally, a reservoir of absorbent material may be juxtaposed with member 20.

After sample addition, it is desirable to add a wash solution to further move the sample away from the reaction zone so that indicator antigen complexes adjacent to the reaction zone are further removed, as shown in FIG. However, in many cases this is not necessary.

For example, as shown in FIG. 4, if the reaction device is equipped with a viewing window because the movement of the gold or indicator system from the reaction area 15 is unclear, it is only necessary to observe the color change in the reaction area 15. A sufficient amount of wash material or sample is added to allow visualization of the clear spot if the sample contains the analyte.

Although FIG. 2 shows a circular shape, it may be of any shape such as a rectangle or a cross. In addition, the reaction area or the sample receiver is not limited to one inlet, and various or multiple inlets may be applied to either or both.
Reactants for detecting various analytes are applied.

The material for the parts is approximately 0°2 to 0.75 micron pore size for the top part and 3-5 microns for the bottom part.
Polyvinyritene fluoride with micron pore size is particularly suitable. A variety of other materials can also be used, such as cellulose acetate, cellulose nitrate, polypropylene, and certain microglass fiber compositions.

The above is based on a competitive binding assay in which the detection target is an antigen, the immunoreagent in the reaction area 15 is an antibody, and the indicator system 18 is gold complexed with an antigen that binds to the antibody. Although this method has been described above, it is also suitable for detecting antigens or antibodies as an analysis target using the Sand-Deutsch method. For example, human chorionic gonadotropin (hC)
In the immunoassay of G), gold conjugated to anti-beta hCG was placed in the bottom layer member of 18 (Fig. 1) and anti-alpha hCG.
The CG (capture Ab) is covalently or otherwise bound to the reaction region 15 of the upper layer member. If the sample contains hCG, the hCG gold/gold body antibody conjugate moves to the reaction zone 15 where the sample hCG portion of the complex binds to the capture antibody (17), resulting in a positive result in the reaction zone 15. It exhibits a red color indicating. If hcc is absent from the sample, there is no hCG to bind into the gold-anti-beta hCG complex. The complex does not bind to capture antibody 17, but instead moves through the reaction zone to a remote location on member 20 and/or member 11 (eg, 12 on member 20).

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view of the device of the present invention taken along the line AA in FIG. 2. FIG. 2 is an embodiment of the device of the invention shown in a circular shape. FIG. 3 shows the device of the present invention after the test liquid has been manipulated and acted upon in the device of FIG. FIG. 4 is a top view of another embodiment of the device of the invention. Description of symbols 10 - one junction device, 11 - second porous material layer, 1
3.13a, 13b, 13cm - liquid barrier means, 1
5-Reaction area, 16-Sample receiving area, 20-First porous material No. 1 B No. 3 B No. 2TB No. 4 Shuite-shrinking amendment 1, Indication of the case 1990 Patent No. 69524 2. Name of the invention Composite device for measuring substances in liquid 3. Relationship with the person making the amendment Patent applicant address Incorporated 4. Agent

Claims (1)

[Claims] 1. A composite device for detecting or measuring the presence of various components in a liquid, comprising at least first and second porous material layers in contact with each other, and one or more samples within the device. a receiving area and a liquid barrier means disposed within said second layer and via said second layer, whereby liquid placed in said sample area is transferred from one member to another. A composite device consisting of a combination of one or more reaction zones communicating with said sample receiving zone through said layer such that said sample receiving zone is transported along said layer. 2. The device of claim 1, wherein the porous material is flat and planar. 3. The device according to claim 2, wherein the porous material is nylon, Teflon, glass fiber, polyvinyl chloride, cellulose acetate, polyvinylidene fluoride, cellulose nitrate or polypropylene. 4. The device of claim 2, wherein each layer is hydrophobic, each layer is hydrophilic, or one layer is hydrophobic and the other layer is hydrophilic. 5. The device of claim 2, wherein the sample receiving area is on the first layer. 6. The device of claim 5, wherein the reaction zone is on the second layer. 7. The device of claim 5, wherein the reaction zone is on the first layer. 8. The device of claim 1, wherein the reaction area is for measuring one or more immunological components of a liquid. 9. The device of claim 8, wherein there are multiple reaction zones. 10. The device according to claim 8, wherein a conjugate of the immunological component to be measured is present in the reaction region. 11. The device according to claim 10, wherein the immunological binding partner is an antibody and the component to be measured is an antigen. 12. In the second layer in the liquid flow path before the reaction zone,
12. The device according to claim 11, wherein an antigen corresponding to the component to be measured is present, and the antigen is conjugated to an indicator system. 13. The device of claim 12, wherein the indicator system is colloidal gold. 14. In the second layer in the liquid flow path before the reaction zone,
12. The device according to claim 11, wherein an antibody that reacts with the antigen to be measured is present, and the antibody is conjugated to an indicator system. 15. The device of claim 14, wherein the indicator system is colloidal gold. 16. The device of claim 1, wherein the barrier is a slot, discontinuity, weld, or compressed area and is positioned within the liquid flow path to adjust the direction and velocity of flow. 17. A reactant is present in a liquid flow path prior to a reaction zone in the device, so that when a sufficient amount of liquid flows along the flow path, the reactant is delivered to the reaction zone. 2. The device of claim 1. 18. The device of claim 16, wherein each layer is protein binding, each layer is protein non-binding, or one layer is protein binding and the other layer is protein non-binding. 19. A device according to claim 16, wherein the layers have the same or different porosity and wicking action.